We have introduced a new methodology using a combination of CyTOF immune profiling, IdU pulse-chasing and mathematical modeling to derive a systemic understanding of immune homeostasis. CyTOF is a recently-acquired instrument (within the NIH-wide Center for Human Immunology) that performs mass cytometry: it is similar to the classical flow cytometer, except that its detection capacity relies on the mass-cytometric resolution of heavy metals tagging antibodies. We have implemented CyTOF within NIH, and demonstrated its capacity to achieve immune profiling with a 40+ panel of antibodies (e.g. targeted against key markers of hematopoietic differentiation). We have also implemented IdU Pulse-chasing, to mark proliferating cells (by incorporation of the metal-tagged nucleotide during DNA replication), and to detect its dilution by cell proliferation or differentiation (by relying on the mass-cytometric capacity of CyTOF): IdU pulse-chasing combines the non-perturbative high-signal noise of BrdU pulse-chasing with the deep immunological profiling of CyTOF. Finally, we developed a mathematical framework to model the experimental results obtained from CyTOF IdU pulse-chasing: our quantitative analysis enables us to animate our network of immune cells, and to quantitate the rates at which cells differentiate We have applied our methodology to delineate the differentiation of NK T cells in the thymus (in collaboration with Dr. Hyun Park from EIB-NCI), of monocytes (in collaboration with Dr. Frederic Geissmann from Memorial Sloan-Kettering) and other systems are under consideration. We are demonstrating the flexibility and resolution power of our new methodology to better analyze the systemic perturbation of hematopoiesis occurring during tumor development (in collaboration with Dr. Romina Goldzmid from CIP-NCI). One major finding so far has been that monocytes of the mouse immune system do not differentiate only in the blood of these animals, but follow a parallel dynamic of differentiation in the bone marrow and in the lymphoid organs. This implies that the main source of blood monocytes may not reside in the blood itself, but rather come from the bone marrow. Such result (obtained without perturbation, at homeostasis) challenges the common understanding of blood monocyte differentiation as obtained from genetic perturbations. A second finding was that NKT2 cells (i.e. Type 2 natural-killer T cells) do not derive from NKT1 cells but rather have a direct (yet inefficient) path of differentiation from immature NK T cells. Again, our kinetic analysis of pulse-chase experiments revealed an alternative path of differentiation that was partially uncovered by genetic perturbation. All in all, this project offers new tools to dissect the dynamics of differentiation and homeostasis in the immune system.